Hyperforin halogenated derivatives, the use thereof and formulations containing them

ABSTRACT

Hyperforin and adhyperforin halogenated derivatives of general formula (I) in which X, R and R 1  have the meanings as defined in the disclosure, the process for the preparation thereof and the use thereof in the pharmaceutical and/or nutritional field, in particular in the treatment of depression, and Alzheimer&#39;s disease.

FIELD OF THE INVENTION

The present invention relates to hyperforin and adhyperforin halogenatedderivatives and the use thereof in the pharmaceutical and/or nutritionalfield, in particular in the treatment of depression and Alzheimer'sdisease.

TECHNOLOGICAL BACKGROUND

Flowering tops of Hypericum perforatum contain a number of classes ofstructurally different substances that act directly or indirectly on thecentral nervous system. The mechanisms of action of these compounds aredifferent and comprise anti-MAO action (Suzuki O R. et al. Planta Med.,272–4, 1984), action on serotonin release and re-uptake (Muller W. E. etal Pharmacopsychiatry, 30, 102–107, 1997) and benzodiazepine-likeactivity (Coot J. M. Pharmacopsychiatry 30,108–112, 1997).

Hyperforin, a floroglucin derivative, is one of the main components ofthe lipophilic fraction of Hypericum perforatum flowering tops; saidfraction also contains adhyperforin, a hyperforin higher homologue,although in lower concentration (Erdelmeier C. A. J.,Pharmacopsychiatry, 31, 2–6, 1998).

Hyperforin has recently been the object of numerous studies thatestablish its important role as an antidepressant (Pharmacopsychiatry,31 Suppl.1, 1–60. 1998). Furthermore, it is recognized that the extractsof Hypericum perforatum can be used for the prophylaxis and treatment ofneurodegenerative diseases, inter alia Alzheimer's disease (WO/9940905,WO0057707). In particular, hyperforin and adhyperforin salts withinorganic cations or ammonium salts were described for this purpose(WO9941220).

It is known from literature that hyperforin is poorly stable in theusual extraction and storage conditions; according to WO 97/13489, thehyperforin content in a St. John's Wort water-alcoholic extract fallsalready after a few weeks. WO 97/13489 further recites that, in order toobtain hyperforin stable extracts, antioxidants should be present duringthe whole work up (extraction, purification and storage). It istherefore evident that the high instability of hyperforin makes thepreparation of hyperforin pharmaceutical formulations quite difficult.In order to obviate to said drawback, compounds more stable thanhyperforin, such as the salts disclosed in WO 99/41220 and thehydroxy-functionalized derivatives (WO 99/64388) cited above, haverecently been prepared.

It is moreover known (Bystrov et al., Bioorg. Khim, 1978) thathyperforin and adhyperforin can be transformed into the correspondingoctahydroderivatives, (IIa and IIb) by catalytic reduction of theisoprene side chains

or into the corresponding tetrahydroderivatives (IIc and IId), byreduction of the keto groups at the 1- and 10-positions to hydroxygroups

DETAILED DISCLOSURE OF THE INVENTION

It has now been found that the compounds obtained by introducing ahalogen at the 8-position of hyperforin, adhyperforin or reductionderivatives thereof possess antidepressant, anxiolytic andanti-neurodegenerative activities surprisingly higher than hyperforinand adhyperforin.

The present invention specifically relates to compounds of formula (I)

wherein X is a halogen atom, R is methyl or ethyl and, alternatively:

a) R₁ is 3-methyl-2-buten-1-yl and oxo groups are present at the 1- and10-positions;

b) R₁ is 3-methyl-but-1-yl and oxo groups are present at the 1- and10-positions;

c) R₁ is 3-methyl-2-buten-1-yl and hydroxy groups are present at the 1-and 10-positions;

d) R₁ is 3-methyl-but-1-yl and hydroxy groups are present at the 1- and10-positions;

and the pharmaceutically acceptable salts or esters thereof.

The term “halogen” herein means fluorine or a halogen selected fromchlorine, bromine and iodine, more preferably chlorine and bromine, mostpreferably chlorine.

Moreover, for the purposes of the present disclosure, “octahydro” meanshyperforin or adhyperforin derivatives in which R₁ is 3-methyl-but-1-yland oxo groups are present at the 1 and 10 positions; “tetrahydro” meanshyperforin or adhyperforin derivatives in which R₁ is3-methyl-2-buten-1-yl and hydroxy groups are present at the 1 and 10positions; “dodecahydro” means hyperforin or adhyperforin derivatives inwhich R₁ is 3-methyl-but-1-yl and hydroxy groups are present at the 1and 10 positions.

Preferred compounds according to the invention are the compounds offormula (I) in which: X is a chlorine or bromine atom, R is methyl orethyl, R₁ is 3-methyl-2-buten-1-yl and oxo groups are present at the 1and 10 positions (in the following defined as: 8-chlorohyperforin Ia,8-chloroadhyperforin Ib, 8-bromohyperforin Ic, 8-bromoadhyperforin Id)

Furthermore, preferred compounds of formula (I) are those wherein: X isa chlorine or bromine atom, R is methyl or ethyl, R₁ is3-methyl-but-1-yl and oxo groups are present at the 1 and 10 positions(in the following defined as: 8-chlorooctahydrohyperforin Ie,8-chlorooctahydroadhyperforin If, 8-bromooctahydrohyperforin Ig,8-bromooctahydroadhyperforin Ih)

Particularly preferred are 8-chlorohyperforin (Ia) and8-chlorooctahydrohyperforin (Ie).

Compounds of formula (I) are prepared by reacting hyperforin,adhyperforin or a tetra-, octa-, dodecahydro derivative thereof with asuitable halogenating agent, preferably with N-chlorosuccinimide orN-bromosuccinimide.

Tetrahydroderivatives (IIa) and (IIb) as mentioned above are obtained byreduction of the keto groups with hydrides, selected for example fromNaBH₄, Redal®, Vitride®, LiAlH₄.

Octahydroderivatives (IIc) and (IId) as above mentioned are obtained byreduction of the isoprene side chains by catalytic hydrogenation, usingfor example palladium on charcoal or Nickel/Raney.

Dodecahydroderivatives (dodecahydrohyperforin IIe anddodecahydroadhyperforin IIf)

are obtained from the octahydroderivatives by treatment with hydrides asindicated above.

Dodecaidroderivatives (IIe) and (IIf) are novel compounds and are alsopart of the present invention.

The process for the preparation of the compounds of the inventionstarting from the flowering tops of Hypericum perforatum can besummarized as follows:

The flowering tops of Hypericum perforatum can be extracted withalcohols or aliphatic ketones, either pure or in a mixture thereof withwater or with gas in supercritical conditions; the resulting extract ispartitioned between n-hexane and aqueous solutions of aliphaticalcohols. The hexane solution is extracted with alkaline methanol toextract hyperforin and adhyperforin. The methanolic solution isacidified, then treated with a weakly basic ion exchange resin, whichselectively retains hyperforin and adhyperforin. The resin is elutedwith acidic methanol and the eluate is concentrated to small volume,then diluted with water and extracted with n-hexane. The hexane solutionis concentrated to small volume and the resulting concentrate is readyfor derivatization. The residue is taken up in chlorinated solvents andthe suitable reactive is added thereto, according to the proceduresreported in the examples.

The present invention further relates to the use of derivatives offormula (I) and the pharmaceutically acceptable salts or esters thereoffor the preparation of medicaments for the therapy of depression, andAlzheimer's disease.

Compounds of formula (I), in particular 8-chloro and 8-bromo hyperforinand 8-chloro and 8-bromo adhyperforin, have shown antidepressant effect.

The antidepressant effect of the compounds of the invention wasevaluated in the rat by the forced swimming test, evaluating theparameters: struggling, floating and swimming according to whatdescribed by Cervo et al. in Neuropharmacology, 26, 14969–72, 1987. Thecompounds were administered in 3 doses: 30 minutes after the pre-test, 5hours and 30 minutes before the test. The results reported in the tablebelow prove that the compounds of the invention are more active thanparent hyperforin.

Struggling Floating Swimming Treatment mg/Kg (sec.) (sec.) (sec.)Carrier  7.0 ± 2.4 174.5 ± 15.9 118.5 ± 15.8 Chlorohyperforin 3.125 46.9± 5.9 72.1 ± 6.7 181.0 ± 11.3 Chlorooctahydro- 6.25 57.3 ± 6.2 63.4 ±9.2 165.6 ± 12.5 hyperforin Hyperforin 6.25 30.4 ± 4.6 60.4 ± 7.3  99.3± 10.6 Desipramin 10 148.3 ± 12.6 53.0 ± 9.2 98.8 ± 7.9

The compounds of the invention also proved particularly active againstAlzheimer's disease, due to their ability to increase APPs, the soluble,harmless form of Alzheimer Precursor Protein (APP). It is in fact knownthat proteolytic cleavage of Alzheimer Precursor Protein (APP) ismediated both by β- and γ-secretase, inducing an increased production ofamyloid peptide Ab1-42 (which also plays a central role in theappearance of Alzheimer's disease), and α-secretase, giving raise tosoluble APPs which have no pathogenic activity (Eslr W. P., Wolfe M. S.,Science, 293, 1449–54, 2001).

The effect of the compounds of the invention on the release of APPsproduced by α-secretase was evaluated in the culture medium of aneuroblastoma cell line (SH-SY5Y) according to the procedure describedby Galbete J. L. et al. in Biochem J. 348, 307–313, 2000.

The results reported in the following table show that the testedcompounds activate α-secretase—mediated APP metabolism, inducing anincrease in APPs secreted in the culture medium:

APPs % Controls 100 10 μM Hyperforin 296 10 μM Chlorohyperforin 627 10μM Chlorooctahydrohyperforin 855

The compounds of the invention can be formulated according toconventional techniques, for example according to what described inRemington's Pharmaceutical Sciences Handbook, XVII Ed. Mack Pub., N.Y.,U.S.A, in the form of soft-gelatin capsules, hard-gelatin capsules,tablets, suppositories; preferably the extract of the invention isformulated in soft-gelatin capsules or in controlled-releaseformulations. The dosage ranges from 10 to 100 mg per unit dose in theusual formulations and up to 200 mg in the controlled-releaseformulations, in this case the suggested dose being 200 mg perdose/daily. Furthermore, the compounds can be administered through thecontrolled-release transdermal route applying the formulation in theproximal area to the cerebral carotid artery derivations. The dosages ofcompound in these formulations range from 10 to 100 mg per dose/daily.

The examples reported hereinbelow illustrate the invention in greaterdetail.

EXAMPLES Example 1 Preparation of Chlorohyperforin

10 kg of flowering tops of Hypericum perforatum and 30 L of methanol areextracted in a 50 L extraction plant and the mass is left to stand atroom temperature for 3 hrs; the extraction is repeated 3 more times,then the combined extracts are concentrated under vacuum to 5 kg and theconcentrate is extracted with 3×5 L of n-hexane. The organic layer isextracted with methanolic KOH until exhaustion of hyperforin andadhyperforin.

This solution is neutralized and filtered through a weakly basicAmberlite resin, which selectively retains hyperforin and adhyperforin;the retained product is eluted again with methanol acidified withphosphoric acid; the methanolic eluate is concentrated under vacuum at25° C., the diluted water and back-extracted with n-hexane untilexhaustion of hyperforin.

The combined organic layers are decolourized with 0.3% charcoal, thendried over Na₂SO₄ and concentrated to an oil below 40° C. under vacuum.After solidification the oil yields a wax (0.52 kg) containing approx.90% of hyperforin.

The residue is taken up in 3 L of CH₂Cl₂ and added with 0.14 kg ofN-chlorosuccinimide, under strong stirring. The solution is left tostand for three hours under stirring at room temperature, checking thedisappearance of hyperforin by TLC using silica gel plates and an-hexane/ethyl acetate 9:1 mixture as eluent (Rf hyperforin 0.20;chlorohyperforin 0.80). After completion of the reaction, 3 L of waterare added; the organic layer is washed with Na₂S₂O₃, then dried overNa₂SO₄. The solvent is evaporated off, the residue is chromatographed onsilica gel eluting with a n-hexane/ethyl acetate 98:2 mixture. Thefractions containing the chloroderivative are concentrated separately,thereby obtaining 0.48 kg of product which, after crystallization frompetroleum ether, has the following chemical-physical and spectroscopicalcharacteristics: [a]_(D)+16 (c=0.5 CH₂Cl₂);

IR ν^(max) (KBr) 1722, 1713, 1446, 1377, 1230, 1064, 831 cm⁻¹;

¹H-NMR (300 MHz CDCl₃): 1.41 (m, H-4), 2.16 (m, H-5), 1.70 (m, H-5′),2.80 (m, H-11), 1.18 (d, J=7 Hz, H-12), 1.02 (d, J=7 Hz, H-13), 1.06 (s,H-14), 2.01 (m, H-15), 1.06 (m, H-15′), 5.03 (m H-17), 1.66 (br s,H-19), 1.60 (br s, H-20), 2.05 (m, H-21), 1.65 (m, H-21′), 4.76 (m,H-22), 1.66 (s, H-24), 1.52 (s, H-25), 3.18 (s, H-26), 4.96 (m, H-27),1.63 (br s, H-29), 1.69 (br s, H-30), 2.60 (m, H-31), 5.17 (dd, J 13.6,H-32), 1.66 (s, H-35).

¹³C-NMR (75 MHz CDCl₃): δ207.6, 205.4, 198.7, 195.9, 139.2, 135.0,134.1, 131.8, 124.5, 121.8, 118.9, 116.8, 85.1, 67.2, 65.1, 56.2, 45.7,40.1, 38.5, 37.5, 31.6, 31.5, 28.2, 26.4, 26.1, 26.0, 25.9, 25.5, 22.2,20.6, 18.6, 18.2, 18.1, 17.9, 13.9.

ESIMS m/z 593, 595 [M+Na⁺] (100.38), 1163, 1165 [2M+Na⁺] (32, 28).

The same chromatographic separation also affords, together with theabove compound, 0.049 kg of chloroadhyperforin having the followingchemical-physical and spectroscopical characteristics:

¹H-NMR (300 MHz CDCl₃): δ5.27–4.75 (4H, m, H-18, H-23, H-28, H-33),2.23, 3.09 (2-H, dd, J=13.4, 8.4 Hz, CH₂-32), 2.63 (2H, m, CH₂-27),2.80–1.42 (10H, m, H-4, H-11, CH₂-5, CH₂-16, CH₂-17, CH₂-22), 1.82–(27H,s, CH₃-20, CH₃-21, CH₃-25, CH₃-26, CH₃-29, CH₃-30, CH₃-31, CH₃-35,CH₃-36), 1.21 (3H, d, J=6.6 Hz, CH₃-14), 0.87 (3H, d, J=6.6 Hz, CH₃-13),1.07 (3H, s, CH₃-15).

¹³C-NMR (75 MHz CDCl₃): δ206.9, 205.4, 198.7, 196.7, 139.2, 135.0,134.1, 131.9, 124.5, 121.8, 118.9, 116.8, 85.1, 67.2, 65.1, 56.2, 46.7,45.7, 45.2, 37.5, 31.6, 31.5, 28.5, 28.2, 26.4, 26.1, 26.0, 25.9, 25.5,18.6, 18.2, 18.1, 18.0, 16.8, 13.9, 11.6.

ESIMS m/z 607, 609 [M+Na⁺] (100, 34), 1191, 1193 [2M+Na⁺] (21, 20).

Example 2 Preparation of Octahydrohyperforin Dicyclohexylammonium Salt

50 g of hyperforin obtained according to what reported in Example 1 aredissolved in 500 ml of ethyl acetate in the presence of 2 g of 5%palladium on charcoal and hydrogenated until complete hydrogenabsorption. The catalyst is filtered off, the hetero-acetic solution isconcentrated to dryness under vacuum and the residue is dissolved inn-hexane. The solution is added with a stoichiometric amount ofdicyclohexylamine, thereby obtaining sufficiently selectivecrystallization of the corresponding salt.

62 g of octahydrohyperforin dicyclohexylammonium salt are obtained,having the following spectroscopical characteristics:

¹H-NMR (300 MHz CDCl₃): δ3.03 (2H, m, CH-DCHA), 2.55–2.30. 2.10–1.76(20H, m, CH₂-DCHA), 1.70–1.10 (22H, m, H-4, H-11, CH₂-5, CH₂-15, CH₂-16,CH₂-17, CH₂-21, CH₂-22, CH₂-26, CH₂-27, CH₂-31, CH₂-32), 0.97–0.83 (24H,d, CH₃-19, CH₃-20, CH₃-24, CH₃-25, CH₃-29, CH₃-30, CH₃-34, CH₃-35),1.19, 1.12 (6H, d, J=6.5 Hz, CH₃-12, CH₃-13), 0.91 (3H, s, CH₃-14).

¹³C-NMR (75 MHz CDCl₃): δ213.1, 211.1, 186.3, 183.6, 119.0, 82.5, 60.8,53.5, 47.5, 44.2, 41.3, 41.0, 40.9, 38.2, 38.1, 37.8, 33.8, 31.0, 30.7,30.0, 29.4, 28.8, 28.3, 27.9, 27.1, 25.4, 25.1, 24.9, 23.5, 23.2, 23.1,22.9, 22.8, 22.7, 22.5, 13.7. ESIMS m/z 567 [M+Na⁺] (100), 1111 [2M+Na⁺](91).

Example 3 Preparation of Chlorooctahydrohyperforin

A solution of 10 g of dicyclohexylammonium octahydrohyperforinate in 60ml of methylene chloride is added with 1.89 g of N-chlorosuccinimide andthe mixture is left under stirring for 30 min. The organic phase isadded with 60 ml of water, washed with a Na₂S₂O₃ saturated solution,then dried over Na₂SO₄; after concentration to dryness the residue ispurified on a silica gel column, eluting the desired compound with anethyl acetate/hexane 95:5 mixuture. The resulting fractions areevaporated to dryness to obtain the desired compound as white powderwhich, after recrystallization from methanol, yields 6.27 g of chloroderivative having the following spectroscopical characteristics:

¹H-NMR (300 MHz CDCl₃): δ3.04–1.04 (22H, m, H-4, H-11, CH₂-5, CH₂-15,CH₂-16, CH₂-17, CH₂-21, CH₂-22, CH₂-26, CH₂-27, CH₃-31, CH₂-32),1.05–0.83 (24H, d, CH₃-19, CH₃-20, CH₃-24, CH₃-25, CH₃-13), CH₃-30,CH₃-34, CH₃-35), 1.19, 1.03 (6H, d, J=6.6 Hz, CH₃-12, CH₃-13). 1–03 (3H,s, CH₃-14).

¹³C-NMR (75 MHz CDCl₃): δ207.6, 205.1, 199.3, 195.8, 84.9, 68.7, 64.5,56.8, 46.1, 43.3, 40.2, 39.9, 38.1, 37.8, 34.8, 33.5, 31.3, 30.7, 29.0,28.8, 28.2, 27.0, 24.8, 23.0, 22.9, 22.8, 22.7, 22.5, 22.4, 22.1, 20.6,14.2.

ESIMS m/z 601, 603 [M+Na⁺] (100.38), 1179, 1181 [2M+Na⁺] (62, 48).

Example 4 Preparation of Dodecahydrohyperforin

1.72 g of dicyclohexylammonium octahydrohyperforinate (M.W.=716; 2.41mmol) are dissolved in 20 ml of THF under magnetic stirring; thesolution is added with a strong excess (3.5 g) of LiAlH₄ (0.092 mol).The progress of the reaction is monitored by TLC (eluent petroleumether/EtOAc 9:1 Rfp=0.6; Rfa=0.6; Rfc=0.52; Rfd=0.18). After ten minutesthe reaction is completed.

The reactive excess is destroyed according to what described in example3. The semisolid reaction mixture is filtered and the residue isthoroughly washed with ethyl acetate. The solution is evaporated todryness, the reaction crude is dissolved in 15 ml of petroleumether/ethyl ethyl 3:1 and the solution is placed in a 150 ml separatoryfunnel. The organic phase is washed three times with 2N sulfuric acidand subsequently with brine. The aqueous phase is removed, the organicone is dried over Na₂SO₄ and concentrated to dryness. The resultingproduct is purified by column chromatography on 75 g of silica gel,eluting the desired compound with petroleum ether/ethyl acetate 99:1.0.9 g of dodecahydrohyperforin are obtained, having the followingphysical-physical and spectroscopical characteristics:

EIMS m/z 548 [M]⁺.

1. A compound of formula (I)

wherein X is a halogen atom, R is methyl or ethyl and, alternatively: a)R₁ is 3-methyl-2-buten-1-yl and oxo groups are present at the 1- and10-positions; b) R₁ is 3-methyl-but-1-yl and oxo groups are present atthe 1- and 10-positions; c) R₁ is 3-methyl-2-buten-1-yl and hydroxygroups are present at the 1- and 10-positions; d) R₁ is3-methyl-but-1-yl and hydroxy groups are present at the 1- and10-positions; and the pharmaceutically acceptable salts or estersthereof.
 2. The compound as claimed in claim 1 wherein X is chlorine orbromine.
 3. The compound as claimed in claim 1 wherein X is chlorine. 4.A compound selected from: 8-chlorohyperforin (Ia), 8-chloroadhyperforin(Ib), 8-bromohyperforin (Ic), 8-bromoadhyperforin (Id)

8-chlorooctahydrohyperforin (Ie), 8-chlorooctahydroadhyperforin (If),8-bromooctahydrohyperforin (Ig), 8-bromooctahydroadhyperforin (Ih)


5. Compounds according to claim 1 for use as medicament.
 6. Apharmaceutical composition containing a compound of claim 1 in mixturewith suitable excipients or carriers.
 7. A pharmaceutical compositioncontaining a compound of claim 2 in mixture with suitable excipients orcarriers.
 8. A pharmaceutical composition containing a compound of claim3 in mixture with suitable excipients or carriers.
 9. A pharmaceuticalcomposition containing a compound of claim 4 in mixture with suitableexcipients or carriers.